Report Expression of Senescence and Apoptosis Biomarkers ...

Case Report

Expression of Senescence and Apoptosis Biomarkers in Syn�\

chronous Bilateral Breast Cancer: A Case Report

Tareq Saleh 1,*, Mohammed El�\Sadoni 2,?, Ahmad Alhesa 2,?, Heyam Awad 2, Mahmoud Jaradat 3,

Mohammad Al�\hazaimeh 3, Rand Dawoud 1, Amel Mryyian 1 and Bilal Azab 2,4

Department of Basic Medical Sciences, Faculty of Medicine, The Hashemite University, Zarqa 13133, Jordan;

1636586@std.hu.edu.jo (R.D.); 1635542@std.hu.edu.jo (A.M.)

2 Department of Pathology, Microbiology and Forensic Medicine, School of Medicine, The University of Jor�\

dan, Amman 11942, Jordan; mhm8191130@ju.edu.jo (M.E.�\S.); ahm8171602@ju.edu.jo (A.A.);

h_awad@ju.edu.jo (H.A.); ba2659@cumc.columbia.edu (B.A.)

3 Department of General Surgery, Jordanian Royal Medical Services, Amman 11942, Jordan; mahmodjara�\

dat@ (M.J.); dr.hazaimeh88.mh@ (M.A.�\h.)

4 Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York,

NY 10032, USA

* Correspondence: tareq@hu.edu.jo; Tel.: +962�\53903333 (ext. 5574)

? Authors contributed equally to this work.

1

Citation: Saleh, T.; El�\Sadoni, M.;

Alhesa, A.; Awad, H.; Jaradat, M.;

Al�\hazaimeh, M.; Dawoud, R.;

Mryyian, A.; Azab, B. Expression of

Senescence and Apoptosis Bi�\

omarkers in Synchronous Bilateral

Breast Cancer: A Case Report. Curr.

Oncol. 2021, 28, 3836�C3845.

�\

Abstract: Background: Synchronous bilateral breast cancer (SBBC) provides a special condition

where two independent breast tumors are exposed to cancer pharmacotherapy within a uniform

pharmacokinetic milieu. Both senescence and apoptosis are established responses to therapy; how�\

ever, they have potentially variable contributions to the overall outcome of treatment, which are yet

to be determined. Methods: In this report, we describe the clinicopathological picture of two SBBC

cases that received standard anticancer treatment and assess their expression profile of several mo�\

lecular hallmarks of senescence and apoptosis. Results: Our analysis identified that synchronous

tumors have variable expression profiles of both senescence�\ and apoptosis�\associated biomarkers,

despite comparable pathological responses to neoadjuvant chemotherapy and current survival

rates. Conclusions: Our results highlight the variable expression of senescence�\ and apoptosis�\asso�\

ciated markers in breast tumors (despite the shared somatic genetic background) and invites a large�\

scale assessment of both senescence and apoptosis in breast cancer tissue in vivo and their contri�\

bution to the pathological response and overall survival.

col28050327

Received: 31 August 2021

Keywords: senescence; apoptosis; BCL�\2; p16INK4a; chemotherapy; synchronous; bilateral; breast

cancer

Accepted: 28 September 2021

Published: 30 September 2021

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Copyright: ? 2021 by the authors. Li�\

censee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and con�\

ditions of the Creative Commons At�\

tribution (CC BY) license (�\

licenses/by/4.0/).

1. Introduction

Synchronous bilateral breast cancer (SBBC) is defined as the diagnosis of two inde�\

pendent primary breast cancer lesions (rather than contralateral metastasis) in both

breasts, within an interval of one month to one year [1,2]. Despite the low frequency of

SBBC (0.2%�C3% of all breast cancers), the incidence of SBBC has been rising due to im�\

proved diagnostic approaches [3,4]. The key risk factors of SBBC include younger age at

presentation [5], a family history of bilateral breast cancer [6], lobular histology [7], multi�\

centricity [8], existence of sclerotic disease [4] and BRCA mutations [9]. Furthermore, an

estrogen receptor (ER)�\positive and human epidermal growth factor receptor 2 (HER2)�\

negative status was reported in the majority of SBBC tumors [10]. The prognosis of SBBC

is largely undetermined, as some evidence supports a worse prognosis [11], while others

suggest that the existence of a synchronous tumor only minimally affects overall survival

Curr. Oncol. 2021, 28, 3836�C3845.

journal/curroncol

Curr. Oncol. 2021, 28

3837

[12]. Nonetheless, the five�\year survival rate for synchronous breast cancer was around

60%, as opposed to 78.7% in metachronous bilateral breast cancer [5].

While the treatment of patients with SBBC is challenging due to the lack of estab�\

lished guidelines specific to the treatment of SBBC [13], many patients receive conven�\

tional neoadjuvant chemotherapy (NAC) as part of their therapy. More importantly, SBBC

provides an avenue whereby two independent tumors, exposed to the same pharmaco�\

logical cancer therapies, exist within the same pharmacokinetic environment. This allows

for the investigation of several cell stress mechanisms that govern the level of pathological

responses to NAC, especially given that a considerable percentage of patients receiving

NAC fail to develop a complete pathological response (pCR) [14].

In this work, we report two cases of SBBC, of distinct tumor biology, with variable

pathological responses to NAC, and utilize their tumor samples to investigate several bi�\

omarkers characteristic of two fundamental cell stress mechanisms: senescence and apop�\

tosis. Apoptosis is the primary form of programmed cell death that mediates the cytotoxic

effects of NAC [15,16] and accounts for the ideal development of pCR following pharma�\

cotherapy. On the other hand, senescence has been increasingly recognized as a funda�\

mental response to anti�\cancer therapies [17], and a potential contributor to unfavorable

therapy outcomes [18]. Interestingly, senescent cells are persistent, and resist apoptosis

via upregulating the pro�\survival proteins (e.g. BCL�\2, BCL�\XL) [19], which, consequently,

renders them sensitive to senolytic agents that target these anti�\apoptotic molecules [20].

Therefore, the interplay between senescence and apoptosis can partly explain the patho�\

logical outcome of a breast tumor following exposure to NAC [21]. Here, we provide evi�\

dence that synchronous breast cancer can have variable expression profiles of both senes�\

cence�\ and apoptosis�\associated hallmarks, despite similar initial pathological responses

to NAC. This report is the first to provide a comprehensive assessment of these bi�\

omarkers in SBBC cases.

2. Case Presentation

The first case was of a 46�\year�\old premenopausal woman who presented to the

breast surgery clinic at King Hussain Medical Center with bilateral breast masses with

left�\sided skin involvement and left axillary lymph node enlargement (Case 1, Table 1).

Her mammography and ultrasonography revealed a dense mass of 3.1 �� 2.4 cm in size,

with irregular margins in the middle outer aspect of the left breast, enlarged pathological

left axillary lymph nodes, and a lobulated mass of 2.4 �� 1.7 cm in size along the upper

outer quadrant of the right breast, which were classified BI�\RADS 4 and 5 for the right

and left breast masses, respectively. The core�\needle biopsy of both breast masses estab�\

lished the diagnosis of invasive ductal carcinoma of the breast (IDC). Moreover, the im�\

munohistochemical analysis of the hormone receptor expression status revealed that the

right�\sided breast tumor was positive for ER+, progesterone receptor (PR+) (100% and

30% expression level, respectively), and positive for HER2+. In contrast, the left�\sided tu�\

mor was positive for both ER+ and PR+ (100% expression level for both), while negative

for HER2�\. Furthermore, fine�\needle aspiration of the left axilla demonstrated the exist�\

ence of malignant cells suggestive of a locally advanced breast carcinoma. However, stag�\

ing studies were negative for evidence of distant metastatic disease. Thus, based on the

8th edition of the American Joint Committee on Cancer (AJCC) staging manual, the clini�\

cal staging was established as follows: stage IIIB (T4bN1M0) for the left�\sided breast tu�\

mor and IIA (T2N0M0) for the right�\sided breast tumor.

Curr. Oncol. 2021, 28

3838

Table 1. Comparison of different diagnostic and treatment characteristics between the two reported

cases.

Case 1

Case 2

Right

Left

Right

Left

BI�\RADS

4

5

5

5

Diagnosis

IDC

IDC

IDC

IDC

Grade

G1

G3

G1

G3

TNM Staging

T2N0M0

T4bN1M0

T2N0M0

T2N2M0

ER+, PR+,

Receptor Status

ER+, PR+, HER2�\ ER+, PR+, HER2+ ER+, PR+, HER2�\

HER2+

NAC Regimen

A+C (4 cycles) followed by D+T+ P A+C (4 cycles) followed by D+T+ P

Bilateral MRM + axillary lymph Bilateral MRM + axillary lymph nodes

Surgical Intervention

nodes dissection

dissection

ypTNM Staging

ypT1c ypN0

ypT1a ypN1a

ypT1a ypN0

pT1b ypN2a

Radiotherapy 50 Gy over 25 frac�\

Radiotherapy 50 Gy over 25 fractions

Adjuvant therapy

tions

trastuzumab and tamoxifen

trastuzumab and tamoxifen

The table describes the clinicopathological characteristics of both tumors, neoadjuvant chemother�\

apy, adjuvant therapy regimens and mode of surgical intervention of both reported cases. The

table also presents the intraoperative pathological staging following the completion of treatment

with NAC. Patients�� information was collected from the databases of King Hussein Medical Cen�\

ter based on the approval by the Institutional Board Review (IRB) committee of the Jordanian

Royal Medical Services (JRMS) No. 6/2021 and in accordance with the World Medical Association

Declaration of Helsinki. Both participating subjects provided informed consent. Abbreviations: BI�\

RADS: Breast Imaging Reporting and Data System; IDC: invasive ductal carcinoma; G: grade; ER:

estrogen receptor; PR: progesterone receptor; HER2: human epidermal growth receptor 2; A: Adri�\

amycin; C: cyclophosphamide; D: docetaxel; T: trastuzumab; P: pertuzumab; MRM: modified radi�\

cal mastectomy; Gy: gray.

The second case was a 54�\year�\old postmenopausal woman who also presented to

the breast clinic at King Hussain Medical Center with bilateral breast masses, associated

with left nipple retraction, peau d��orange skin, and palpable axillary lymph nodes (Case

2, Table 1). Her mammography and ultrasonography revealed an irregular mass of 5x3.7

cm in size containing microcalcifications, resulting in tissue distortion and skin thickening

in the upper outer quadrant of the left breast. Moreover, ultrasonography showed en�\

larged suspicious lymph nodes in the left axilla, and multiple ill�\defined densities (the

largest was 2.5x1.5 cm) with tiny calcifications in the lower inner quadrant of the right

breast, categorized as BIRADS 5 in both breasts. The core�\needle biopsy confirmed the

diagnosis of bilateral IDC, whereas nodal metastasis was detected by FNA of the left ax�\

illa. The receptor status of the right�\sided tumor was ER+, PR+ (100% for both), and

HER2+. In comparison, the left�\sided tumor exhibited lower expression of ER+ and PR+

(50% and 30%, respectively), and was negative for HER2�\. No evidence of distant metas�\

tasis was detected. Accordingly, clinical staging based on the 8th AJCC staging manual

was established as follows: IIIA (T2N2M0) for the left�\sided breast tumor and IIA

(T2N0M0) for the right�\sided breast tumor.

Following the discussion of the treatment strategy by the onco�\surgical multidisci�\

plinary team, both patients received four cycles of NAC, namely, adriamycin + cyclophos�\

phamide followed by docetaxel, trastuzumab, and pertuzumab. Two months following

the completion of pharmacotherapy, Case 1 underwent a bilateral modified radical mas�\

tectomy (MRM) and bilateral axillary lymph node dissection. The histopathology of the

right�\sided tumor showed a residual invasive tumor of 17 mm in size, grade 2, 17 axillary

lymph nodes negative for metastasis, no lympho�\vascular or perineural invasion, and the

receptor status was ER+, PR+, and HER2+ (ypT1c ypN0) (Table 1). The left side showed a

tiny residual focus of an invasive tumor 2 mm in size, grade 2, two lymph nodes positive

for metastasis out of 13, positive lympho�\vascular and not identified perineural invasion,

Curr. Oncol. 2021, 28

3839

and the receptor status was ER+ and PR+ and HER2�\ (ypT1a ypN1a). On the other hand,

Case 2 underwent bilateral MRM with axillary lymph node dissection, three months fol�\

lowing the completion of pharmacotherapy. The surgical pathology report indicated the

following: 1) right breast: residual tumor of 3 mm in size, grade 1, 22 axillary lymph nodes

were negative for metastasis, no lymphovascular or perineural invasion, and the receptor

status was ER+, PR+ (100% for both), and HER 2+ (ypT1a YpN0) (Table 1); 2) left breast:

residual tumor of 7 mm in size, grade 3, 7 lymph nodes were positive for metastasis out

of 13, lymphovascular and perineural invasion was detected, and the receptor status was

ER+ and PR+ (50% and 30%, respectively), but was negative for HER2�\ (ypT1b YpN2a).

Based on the receptor status, both patients were commenced on postoperative

trastuzumab and tamoxifen to decrease the risk of recurrence. Moreover, within three

months of surgical intervention, both patients also received adjuvant radiotherapy with

50 Gy over 25 fractions (Table 1). To date (16 months and 11 months after treatment initi�\

ation for Case 1 and Case 2, respectively), both patients have remained asymptomatic,

without clinical or radiological evidence of breast cancer recurrence.

3. Biochemical Analysis

We then wanted to look at the expression level of several biomarkers associated with

both senescence and apoptosis, aiming to identify variabilities in their expression level in

SBBC, and whether it is linked to tumor response to therapy. For that, we employed im�\

munohistochemistry techniques to detect and quantify the expression of these protein

markers using true�\cut and core�\needle biopsy tumor (T) blocks for both cases, as de�\

scribed previously [22] (Figure 1). For comparison, we also provided an assessment of

protein expression levels in normal breast epithelium (N) for each corresponding breast

of both cases (Figure 1). The examined senescence�\associated markers included: p16INK4a,

p21Cip1, Ki67, Lamin B1, histone 3 lysine 9 tri�\methylated (H3K9Me3) and vascular endo�\

thelial growth factor (VEGF) [23], while the examined apoptosis markers were: p53,

NOXA, MCL�\1, BAX, BCL�\XL [24].

For senescence, in Case 1, the right tumor was positive for p21Cip1 (T: 60%, N: 80%),

Ki67 (T: 35%, N: negative), Lamin B1 (T: 60%, N: 80%), and H3K9Me3 (T: 60%, N: 90%)

(Table 2). Similarly, the left tumor was positive for p21Cip1 (T: 5%, N: 50%), Ki67 (T: 65%,

N: negative), Lamin B1 (T: 50%, N: 10%), and H3K9Me3 (T: ................
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